ABCMdb

ABCA1 p.Leu222Ala

None has been submitted yet.

Publications

PMID: 21209084 [PubMed] Ohnsorg PM et al: "Carboxyl terminus of apolipoprotein A-I (ApoA-I) is necessary for the transport of lipid-free ApoA-I but not prelipidated ApoA-I particles through aortic endothelial cells."

No. Sentence Comment

46 Generation of Adenoviruses Expressing ApoA-I(L218A/L219A/ V221A/L222A)-The apoA-I gene lacking the BglII restriction site (that is present at nucleotide positions 181 of the genomic sequence relative to the ATG codon of the gene) was cloned into the pcDNA3.1 vector to generate the pcDNA3.1- apoA-I(⌬BglII) plasmid as described (30). Login to comment
47 This plasmid was used as a template to introduce the apoA-I(L218A/L219A/ V221A/L222A) mutations in apoA-I using the mutagenesis kit QuikChange௡ XL (Stratagene) and the mutagenic primers. Login to comment
56 The mutant protein was isolate from the culture medium of HTB-13 cells infected with the apoA-I(L218A/ L219A/V221A/L222A)-expressing adenovirus as described (22, 26). Login to comment
94 The two mutants with a defective carboxyl-terminal sequence, apoA-I(⌬185-243) and apoA-I(L218A/L219A/V221A/L222A), showed 50 and 80% decreases in total and specific endothelial binding, respectively (Fig. 1). Login to comment
103 The results are represented as means Ϯ S.D. (error bars) of at least three individual experiments. Transendothelial ApoA-I Transport Is a Two-step Process 7746 binant WT apoA-I, and apoA-I(⌬144-165) did not differ from each other whereas the apoA-I(⌬185-243) and apoA-I(L218A/ L219A/V221A/L222A) mutants showed strongly reduced total and specific cell association, and the apoA-I(⌬1-59) and apoA-I(⌬1-59/⌬185-243) mutants showed massively increased total cell association which could not be competed by WT apoA-I (data not shown). Login to comment
109 In contrast, the specific transports of apoA-I(⌬185-243) and apoA-I(L218A/L219A/V221A/L222A) were decreased by 90%. Login to comment
112 Therefore, we compared the particle sizes of WT apoA-I and the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant before and after transport through ECs (Fig. 3). Login to comment
114 By contrast, the comparison of the gel filtration profiles of the binding- and transport-defective apoA-I(L218A/ L219A/V221A/L222A) mutant before and after incubation with ECs did not reveal the occurrence of any new peak (Fig. 3B). Login to comment
116 The larger sized fraction of apoA-I(L218A/L219A/V221A/L222A) has a higher elution volume than the fraction formed after transport of WT apoA-I and did not change after transport. Login to comment
117 We therefore assume that this fraction represents lipid-free aggregates of apoA-I(L218A/ L219A/V221A/L222A). Login to comment
138 Binding and Transport of Reconstituted HDL Containing WT ApoA-I or ApoA-I(L218A/L219A/V221A/L222A)-Next, we exploited the apoA-I(L218A/L219A/V221A/L222A) mutant with defects in ABCA1-mediated lipid efflux as well as specific endothelial binding and transport to test the hypothesis that transendothelial transport of lipid-free apoA-I occurs by a two-step mechanism in which apoA-I is first lipidated by ABCA1-dependent lipid efflux to then undergo ABCA1-independent transport through ECs. Login to comment
139 We first lipidated WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment
141 Both WT apoA-I and apoA-I(L218A/L219A/V221A/L222A) formed particles that had a higher electrophoretic mobility than the respective lipid-free apolipoproteins. Login to comment
142 rHDL containing apoA-I(L218A/L219A/ V221A/L222A) were slightly less negatively charged than rHDL containing WT apoA-I (Fig. 6A). Login to comment
143 In addition, the Sudan Black staining of the agarose gel clearly reveals the lipidation of both WT apoA-I and apoA-I(L218A/L219A/V221A/L222A). Login to comment
145 Electron microscopy revealed that both WT apoA-I and apoA-I(L218A/L219A/ V221A/L222A) formed discoidal particles (Fig. 6C). Login to comment
147 Neither the specific binding (Fig. 6D) nor the specific transport (Fig. 6E) differed between particles containing either WT apoA-I or the mutant apoA-I(L218A/ L219A/V221A/L222A). Login to comment
148 Thus prelipidation can overcome the binding and transport defects of the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant. Login to comment
154 Particle size of WT apoA-I and apoA-I(L218A/L219A/V221A/L222A) before and after transport through ECs. Login to comment
158 B, 125 I-apoA-I(L218A/L219A/V221A/L222A). Login to comment
166 In contrast to apoA-I(L218A/L219A/V221A/L222A), prelipidation of either apoA-I(⌬1-59) or apoA-I(⌬1-59/⌬185-243) did not rescue their FIGURE 4. Login to comment
178 Binding and transport of rHDL containing WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment
181 D, specific binding at 4 °C of 10 ␮g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) was determined. Login to comment
182 E, specific transport of 10 ␮g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) through a monolayer of ECs was determined. Login to comment
187 down of ABCG1 and SR-BI but not of ABCA1 decreased the specific transendothelial transport of prelipidated WT apoA-I (Fig. 8A) and prelipidated apoA-I(L218A/L219A/V221A/ L222A) (Fig. 8B). Login to comment
188 The reduction of the transport capacity of lipidated apoA-I(L218A/L219A/V221A/L222A) by knockdown of ABCG1 or SR-BI was smaller (-44% Ϯ7%) compared with lipidated WT apoA-I (-60% Ϯ 15%). Login to comment
194 Also, in our endothelial transwell cell culture model, both apoA-I(⌬185-243) and apoA-I(L218A/L219A/V221A/L222A) failed to form HDL-like particles although only the specific but not the nonspecific fraction of transendothelial transport was abolished. Login to comment
207 The formation of a high affinity complex of apoA-I with ABCA1 is thought to play FIGURE8.RoleofABCA1,ABCG1,andSR-BIinthetransportofrHDLcontainingWTapoA-IorapoA-I(L218A/L219A/V221A/L222A).ECsweretransfected with siRNA coding for ABCA1, ABCG1, SR-BI and not coding siRNA. Login to comment
211 B, specific transport of rHDL containing apoA-I(L218A/ L219A/V221A/L222A). Login to comment
218 The apoA-I(L218A/L219A/V221A/L222A) mutant, which shows strongly reduced specific but normal nonspecific transendothelial transport (data not shown), was not recovered as a lipidated particle (Fig. 3). Login to comment
221 These downstream pathways may be shared with the transport of HDL because knockdown of ABCG1 or SR-BI inhibited the specific transport of native HDL (15) as well as rHDL containing either WT apoA-I or apoA-I(L218A/L219A/ V221A/L222A) (Fig. 8). Login to comment
222 In agreement with normal binding of rHDL containing apoA-I(L218A/L219A/V221A/L222A), ldlA-7 cells expressing SR-BI were previously found to bind rHDL containing apoA-I(⌬185-243) with affinity similar to that of rHDL with WT apoA-I (46). Login to comment
45 Generation of Adenoviruses Expressing ApoA-I(L218A/L219A/ V221A/L222A)-The apoA-I gene lacking the BglII restriction site (that is present at nucleotide positions 181 of the genomic sequence relative to the ATG codon of the gene) was cloned into the pcDNA3.1 vector to generate the pcDNA3.1- apoA-I(èc;BglII) plasmid as described (30). Login to comment
55 The mutant protein was isolate from the culture medium of HTB-13 cells infected with the apoA-I(L218A/ L219A/V221A/L222A)-expressing adenovirus as described (22, 26). Login to comment
92 The two mutants with a defective carboxyl-terminal sequence, apoA-I(èc;185-243) and apoA-I(L218A/L219A/V221A/L222A), showed 50 and 80% decreases in total and specific endothelial binding, respectively (Fig. 1). Login to comment
101 The results are represented as means afe; S.D. (error bars) of at least three individual experiments. Transendothelial ApoA-I Transport Is a Two-step Process 7746 binant WT apoA-I, and apoA-I(èc;144-165) did not differ from each other whereas the apoA-I(èc;185-243) and apoA-I(L218A/ L219A/V221A/L222A) mutants showed strongly reduced total and specific cell association, and the apoA-I(èc;1-59) and apoA-I(èc;1-59/èc;185-243) mutants showed massively increased total cell association which could not be competed by WT apoA-I (data not shown). Login to comment
107 In contrast, the specific transports of apoA-I(èc;185-243) and apoA-I(L218A/L219A/V221A/L222A) were decreased by 90%. Login to comment
110 Therefore, we compared the particle sizes of WT apoA-I and the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant before and after transport through ECs (Fig. 3). Login to comment
115 We therefore assume that this fraction represents lipid-free aggregates of apoA-I(L218A/ L219A/V221A/L222A). Login to comment
136 Binding and Transport of Reconstituted HDL Containing WT ApoA-I or ApoA-I(L218A/L219A/V221A/L222A)-Next, we exploited the apoA-I(L218A/L219A/V221A/L222A) mutant with defects in ABCA1-mediated lipid efflux as well as specific endothelial binding and transport to test the hypothesis that transendothelial transport of lipid-free apoA-I occurs by a two-step mechanism in which apoA-I is first lipidated by ABCA1-dependent lipid efflux to then undergo ABCA1-independent transport through ECs. Login to comment
137 We first lipidated WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment
140 rHDL containing apoA-I(L218A/L219A/ V221A/L222A) were slightly less negatively charged than rHDL containing WT apoA-I (Fig. 6A). Login to comment
146 Thus prelipidation can overcome the binding and transport defects of the dysfunctional apoA-I(L218A/L219A/V221A/L222A) mutant. Login to comment
152 Particle size of WT apoA-I and apoA-I(L218A/L219A/V221A/L222A) before and after transport through ECs. Login to comment
156 B, 125 I-apoA-I(L218A/L219A/V221A/L222A). Login to comment
164 In contrast to apoA-I(L218A/L219A/V221A/L222A), prelipidation of either apoA-I(èc;1-59) or apoA-I(èc;1-59/èc;185-243) did not rescue their FIGURE 4. Login to comment
176 Binding and transport of rHDL containing WT apoA-I or apoA-I(L218A/L219A/V221A/L222A). Login to comment
179 D, specific binding at 4 &#b0;C of 10 òe;g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) was determined. Login to comment
180 E, specific transport of 10 òe;g/ml 125 I-rHDL particles containing either WT apoA-I or apoA-I(L218A/L219A/V221A/L222A) through a monolayer of ECs was determined. Login to comment
185 Transendothelial ApoA-I Transport Is a Two-step Process MARCH 11, 2011ߦVOLUME 286ߦNUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY down of ABCG1 and SR-BI but not of ABCA1 decreased the specific transendothelial transport of prelipidated WT apoA-I (Fig. 8A) and prelipidated apoA-I(L218A/L219A/V221A/ L222A) (Fig. 8B). Login to comment
186 The reduction of the transport capacity of lipidated apoA-I(L218A/L219A/V221A/L222A) by knockdown of ABCG1 or SR-BI was smaller (afa;44% afe;7%) compared with lipidated WT apoA-I (afa;60% afe; 15%). Login to comment
192 Also, in our endothelial transwell cell culture model, both apoA-I(èc;185-243) and apoA-I(L218A/L219A/V221A/L222A) failed to form HDL-like particles although only the specific but not the nonspecific fraction of transendothelial transport was abolished. Login to comment
205 The formation of a high affinity complex of apoA-I with ABCA1 is thought to play FIGURE8.RoleofABCA1,ABCG1,andSR-BIinthetransportofrHDLcontainingWTapoA-IorapoA-I(L218A/L219A/V221A/L222A).ECsweretransfected with siRNA coding for ABCA1, ABCG1, SR-BI and not coding siRNA. Login to comment
209 B, specific transport of rHDL containing apoA-I(L218A/ L219A/V221A/L222A). Login to comment
216 The apoA-I(L218A/L219A/V221A/L222A) mutant, which shows strongly reduced specific but normal nonspecific transendothelial transport (data not shown), was not recovered as a lipidated particle (Fig. 3). Login to comment
219 These downstream pathways may be shared with the transport of HDL because knockdown of ABCG1 or SR-BI inhibited the specific transport of native HDL (15) as well as rHDL containing either WT apoA-I or apoA-I(L218A/L219A/ V221A/L222A) (Fig. 8). Login to comment
220 In agreement with normal binding of rHDL containing apoA-I(L218A/L219A/V221A/L222A), ldlA-7 cells expressing SR-BI were previously found to bind rHDL containing apoA-I(èc;185-243) with affinity similar to that of rHDL with WT apoA-I (46). Login to comment

PMID: 23990662 [PubMed] Fotakis P et al: "Role of the hydrophobic and charged residues in the 218-226 region of apoA-I in the biogenesis of HDL."

No. Sentence Comment

7 Adenovirus-mediated gene transfer of apoA-I[L218A/ L219A/V221A/L222A] in apoA-I d1a;/d1a; mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-beta-and ॷ4-HDL particles. Login to comment
21 Published, JLR Papers in Press, August 29, 2013 DOI 10.1194/jlr.M038356 Role of the hydrophobic and charged residues in the 218-226 region of apoA-I in the biogenesis of HDL 1 Panagiotis Fotakis,* ,ߤ Andreas K. Kateifides,* ,ߤ Christina Gkolfinopoulou, &#a7; Dimitra Georgiadou, &#a7; Melissa Beck,* ,ߤ Katharina Gr&#fc;ndler,* Angeliki Chroni, &#a7; Efstratios Stratikos, &#a7; Dimitris Kardassis, ߤ and Vassilis I. Zannis 2, * Whitaker Cardiovascular Institute,* Boston University School of Medicine, Boston, MA 02118; Department of Biochemistry,ߤ University of Crete Medical School, Heraklion, Crete, Greece 71110; and National Center for Scientific Research "Demokritos",&#a7; Athens, Greece 15310 Abbreviations: ANS, 8-anilino-1-naphthalene-sulfonate; apoA-I[218-222], the apoA-I carrying the L218A/L219A/V221A/L222A mutations; apoA-I[E223A/K226A], the apoA-I carrying the E223A/K226A mutations apoA-I afa;/afa; , apoA-I-deficient; apoA-I afa;/afa; &#d7; apoE afa;/afa; , apoA-I and apoE double-deficient; CD, circular dichroism; cpt-cAMP, 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphatase; DMPC, dimyristoyl-L-ॷ- phosphatidylcholine; EM, electron microscopy; FPLC, fast-protein liquid chromatography; GdnHCl, guanidine hydrochloride; HEK293, human embryonic kidney 293; HTB-13, SW 1783 human astrocytoma; pfu, plaque-forming unit; POPC, beta-oleoyl-ॹ-palmitoyl-L-ॷ-phosphatidylcholine; rHDL, reconstituted HDL; WMF, wavelength of maximum fluorescence; WT, wild-type. Login to comment
60 In addition to the drastic effect of the L218A/L219A/ V221A/L222A mutations on the biogenesis of HDL, the mutants also inhibited the ability of lipid-free apoA-I to promote transendothelial transport (20), as well as its bactericidal activity against Gram-negative bacteria (21), indicating the importance of the 218-222 residues for the functions of apoA-I. Login to comment
74 The L218A/L219A/V221A/L222A mutations in apoA-I resulted in a great increase in plasma apoA-IV that floated in the IDL/LDL/HDL2/HDL3 region (Fig. 3D and supplementary Fig. II) and the presence of apoB-48 in the HDL region (Fig. 3E). Login to comment
132 Effect of the L218A/L219A/V221A/L222A and E223A/ K226A mutations on the ॷ-helical content, thermal unfolding, chemical unfolding, and hydrophobic surface exposure of apoA-I To test whether the functional changes of the two apoA-I mutants are accompanied by changes in the structure and conformation of the protein, we used an array of biophysical assays to evaluate the effects of these mutations (Fig. 6). Login to comment
142 L218A/L219A/V221A/L222A and E223A/K226A mutations alter the functional and physicochemical properties of apoA-I The in vitro experiments showed that, compared with WT apoA-I, the capacity of the apoA-I[218-222] mutant to promote ABCA1-mediated cholesterol efflux and to activate LCAT was 20% and 66%, respectively. Login to comment
173 Calculated biophysical parameters for WT and mutant apoA-I forms Mutation Circular Dichroism Thermal Denaturation Chemical Denaturation ANS Binding ApoA-I ॷ-Helix Tm (&#b0;C) Slope Cooperativity Index (n) èc;H (kcal/mol) èc;GD o (kcal/mol) D1/2 (M) mc (kcal mol afa;2 ) Fold Increasea WT 60.0 &#b1; 1.5 55.6 &#b1; 0.4 8.3 &#b1; 0.4 6.4 &#b1; 0.2 25.9 &#b1; 1.5 6.3 &#b1; 0.4 1.01 &#b1; 0.02 6.3 &#b1; 0.4 6.0 &#b1; 0.4 L218A/L219A/V221A/L222A 52.7 &#b1; 1.6b 56.1 &#b1; 0.8 4.6 &#b1; 0.2 b 9.7 &#b1; 0.6 b 46.4 &#b1; 2.4 b 6.6 &#b1; 0.4 1.00 &#b1; 0.03 6.4 &#b1; 0.4 3.5 &#b1; 0.2 b E223A/K226A 55.8 &#b1; 1.1 c 53.4 &#b1; 0.2 b 10.4 &#b1; 1.4 d 5.3 &#b1; 0.7 b 21.01 &#b1; 2.2 e 2.5 &#b1; 0.2 b 0.88 &#b1; 0.02 f 2.8 &#b1; 0.1 b 9.6 &#b1; 0.5 b Values are means &#b1; SD from three or four experiments. Login to comment

PMID: 25522986 [PubMed] Zannis VI et al: "HDL biogenesis, remodeling, and catabolism."

No. Sentence Comment

76 Adenovirus-mediated gene transfer of these mutants in apoA-I / x apoE / mice (Fotakis et al. 2013a, b) showed that compared to the WT apoA-I, the expression of an apoA-I[L218A/L219A/V221A/L222A] mutant decreased plasma cholesterol, apoA-I, and HDL cholesterol levels and generated preb2;- and b1;4 HDL subpopulations (Fotakis et al. 2013a) (Table 1 and Fig. 3a-g). Login to comment
77 To eliminate the involvement of apoE in the generation of apoE-containing HDL particles (Kypreos and Zannis 2007), the apoA-I[L218A/L219A/V221A/L222A] mutant was expressed in apoA-I / x apoE / mice via adenovirus-mediated gene transfer (Fotakis et al. 2013a). Login to comment
81 Table 1 Plasma lipids and hepatic mRNA levels of apoA-I / or apoA-I / x apoE / mice expressing WT and mutant forms of apoA-I in the presence and absence of LCAT as indicated Protein expressed Total cholesterol (mg/dL) Triglycerides (mg/dL) Relative apoA-I mRNA (%) apoA-I plasma levels (mg/dL) WT apoA-I in apoA-I / mice 278 74 78 24 100 26 260 40 apoA-I[L218A/L219A/V221A/ L222A] in apoA-I / mice 45 14 50 20 95 24 41 5 WT apoA-I in apoA-I / x apoE / mice 1,343 104 294 129 100 13 - apoA-I[L218A/L219A/V221A/ L222A] in apoA-I / x apoE / mice 778 52 18 2 92 23 - apoA-I[L218A/L219A/V221A/ L222A] plus LCAT in apoA-I / x apoE / mice 754 122 37 10 90 3 - Fig. 3 (a-g) ApoA-I mutations and their effect on the HDL phenotypes. Login to comment
82 Analysis of plasma of apoA-I / x apoE / mice infected with adenoviruses expressing the WT apoA-I or the apoA-I [L218A/L219A/V221A/L222A] mutant, as indicated, by density gradient ultracentrifugation and SDS-PAGE (a, e). Login to comment
83 EM analysis of HDL fractions 6-7 obtained from apoA-I / x apoE / mice expressing the WT apoA-I or the apoA-I[L218A/L219A/V221A/L222A] mutant, as indicated, following density gradient ultracentrifugation of plasma (b, f). Login to comment
85 Two-dimensional gel electrophoresis of plasma of apoA-I / x apoE / mice infected with adenoviruses expressing WT apoA-I or the apoA-I[L218A/L219A/V221A/L222A] mutant, as indicated (d, g) These particles may originate from apoB-48 containing lipoproteins that are found in the HDL fractions (Fig. 3c). Login to comment
88 Co-expression of the apoA-I[L218A/L219A/V221A/L222A] mutant and human LCAT in apoA-I / x apoE / mice did not correct the plasma apoA-I levels and did not correct the aberrant HDL phenotype. Login to comment
92 The phenotype produced by the apoA-I[L218A/L219A/V221A/L222A] mutations is distinct from all previously described phenotypes and cannot be corrected by overexpression of LCAT. Login to comment
94 Although other interpretations are possible, the in vivo and in vitro data suggest that the interaction of the apoA-I [L218A/L219A/V221A/L222A] mutant with ABCA1 results in defective lipidation of apoA-I that leads to the generation of preb2;-HDL particles that are not a good substrate for LCAT. Login to comment
99 The phenotype generated by the expression of the apoA-I[F225A/V227A/ F229A/L230A] mutant was similar to that obtained with the apoA-I[L218A/ L219A/V221A/L222A] mutant. Login to comment
421 ApoA-I mutants with defective C-terminal apoA-I[Ɗ(185-243)] and apoA-I[L218A/L219A/V221A/ L222A] had 80 % decreased specific binding and 90 % decreased specific transport by aortic endothelial cells. Login to comment